Antibodies of symptomatic human immunodeficiency virus type 1-infected individuals are directed to the V3 domain of noninfectious and not of infectious virions present in autologous serum.

The present study was designed to determine the antibody specificity for the human immunodeficiency virus type 1 (HIV-1) V3 domains of infectious and noninfectious virions present in the serum of AIDS patients. To accomplish this, HIV-1 was isolated in the presence of autologous antibodies from the serum samples of six AIDS patients in HIV-1-negative donor peripheral blood mononuclear cells by short-term cultivation. The isolated virus, defined as the infectious cell-free virus (iCFV), was characterized by sequence analysis of the proviral DNA coding for the third hypervariable (V3) region of the external glycoprotein gp120. This was carried out by amplifying and cloning the V3 region. In all six cases studied, 20 randomly selected V3 clones derived from the proviral DNA of the iCFV, 20 clones from patient cell-free virus, and 20 clones from cell-integrated virus were sequenced to study the distribution and frequency of the intrapatient virus population. The number of major virus variants in the six patients ranged from three to nine. The various V3 sequences found in the AIDS patients showed the typical amino acid pattern of the syncytium-inducing and non-syncytium-inducing viral phenotypes characteristic for the late stage of infection. However, only one patient-specific iCFV variant was detected within the 20 V3 clones analyzed per virus isolation. For the six patients a total of 34 V3-loop variants, either iCFV or non-iCFV, was observed. All 34 V3-loop sequences were expressed as glutathione-S-transferase fusion proteins (V3-GST). The autologous antibody response to the V3-GST fusion proteins was studied by Western immunoblot analysis. A strong antibody response to almost all non-iCFV V3-GST proteins was found in the sera of the six patients. In contrast, the autologous antibody response to the six iCFV V3 loops was undetectable (in four patients) or very faint (in two patients) compared with that to the non-iCFV V3 loops. Five of the six iCFV loops showed positively charged amino acids at positions strongly associated with the syncytium-inducing phenotype. These findings suggest that our in vitro isolation system selects for virions which are not recognized by V3-specific antibodies and are infectious both in vitro and in vivo.     

NAD(P)H Dehydrogenase-Dependent Cyclic Electron Flow around Photosystem I in the Cyanobacterium Synechocystis PCC 6803: a Study of Dark-Starved Cells and Spheroplasts

Electron donation to P700⁺ through plastoquinone in the intersystemchain from both respiratory substrates and the photoreductantsin PSI has been shown to be mediated by the NAD(P)H-dehydrogenasecomplex (NDH) in Synechocystis PCC 6803 cells [Mi et al. (1992)Plant Cell Physiol. 33: 1233]. To confirm the participationof NDH in the cyclic electron flow around PSI, the redox kineticsof P700 and Chi fluorescence were analyzed in cells rendereddeficient in respiratory substrates by dark starvation and inspheroplasts. Dark-starved cells showed a high steady-state level of P700⁺under far-red (FR) illumination and the plastoquinone pool wasin a highly oxidized state. An NDH-defective mutant consistentlyshowed a high level of P700 oxidation under FR before and afterthe dark starvation. Donation of electrons either from exogenousNADPH or from photoreduced NADPH⁺ to the intersystem chain viaplastoquinone was demonstrated using spheroplasts from wild-typecells, but not those from the NDH-defective mutant, as monitoredby following changes in the kinetics of Chi fluorescence andthe redox state of P700. The electron flow to PSI via plastoquinone,mediated by NADPH, was sensitive to rotenone, Hg²⁺ ions and2-thenoyltrifluoroacetone, inhibitors of mitochondrial NDH andsuccinate dehydrogenase, but not to antimycin A. The pool sizeof electrons that can be donated to P700⁺ from the cytosol throughthe intersystem chain increased with increasing duration ofillumination time by actinic light and was sensitive to rotenonein both wild-type cells and spheroplasts, but no such resultswere obtained in the NDH-defective mutant of Synechocystis 6803.The results support our previous conclusion that NDH is a mediatorof both respiratory electron flow and cyclic electron flow aroundPSI to the intersystem chain in the cyanobacterium Synechocystis. (Received August 20, 1993; Accepted November 22, 1993)     

On the relationship between chlorophyll fluorescence quenching and the quantum yield of electron transport in isolated thylakoids

The relationship between the empirical fluorescence index F/Fm and the quantum yield of linear electron flow, ^s, was investigated in isolated spinach thylakoids. Conditions were optimised for reliable determination of F/Fm and ^s with methyl viologen or ferricyanide as electron acceptors under coupled and uncoupled conditions. Ascorbate in combination with methyl viologen was found to stimulate light-induced O₂-uptake which is not reflected in F/Fm and interpreted to reflect superoxide reduction by ascorbate. In the absence of ascorbate, the plot of F/Fm vs. ^s was mostly linear, except for the range of high quantum yields, i.e. at rather low photon flux densities. With ferricyanide as acceptor, use of relatively low concentrations (0.1–0.3 mM) was essential for correct Fm-determinations, particularly under uncoupled conditions. Under coupled and uncoupled conditions the same basic relationship between F/Fm and ^s was observed, irrespective of ^s being decreased by increasing light intensity or by DCMU-addition. The plots obtained with methyl viologen and ferricyanide as acceptors were almost identical and similar to corresponding plots reported previously by other researchers for intact leaves. It is concluded that the index F/Fm can be used with isolated chloroplasts for characterisation of such types of electron flow which are difficult to assess otherwise, as e.g. O₂ dependent flux. The origin of the non-linear part of the relationship is discussed. An involvement of inactive PS II centers with separate units and inefficient Q_A-Q_B electron transfer is considered likely.Abbreviations AsA - ascorbate - DCMU - 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MDA - monodehydroascorbate - MV - methyl viologen - PAR - photosynthetically active radiation - SOD - superoxide dismutase This paper is dedicated to David Walker who after 40 years in the field of photosynthesis is now retiring from his duties at Sheffield University.     

Inhibition by ethoxyzolamide of a photoacoustic uptake signal in leaves: Evidence for carbonic anhydrase catalyzed CO2-solubilisation

A photoacoustic pulse-modulation technique is applied for the study of a CO₂-stimulated gas uptake signal in leaves (Reising and Schreiber, Photosynthe Res 31: 227–238, 1992). It is shown that this uptake signal can be substantially suppressed by application of the carbonic anhydrase inhibitor, ethoxyzolamide, to leaf discs. This inhibitor does not affect the O₂-evolution signal in air or the chlorophyll fluorescence induction pattern at high CO₂, when non-saturating light intensities are used. On the basis of these findings it is concluded that at least a major part of the CO₂-stimulated photoacoustic uptake signal results from light-modulated CO₂-solubilisation catalysed by carbonic anhydrase. Modulated CO₂-solubilisation appears likely to be induced by light driven H⁺-translocation from the stroma into the thylakoid lumen. Comparison of the induction patterns of chlorophyll fluorescence quenching and the uptake signal suggests a correlation between membrane energisation and CO₂-uptake. The importance of O₂-dependent electron flow as a major cause of membrane energisation is discussed. It is proposed that in the absence of CO₂ the combination of Mehler- and ascorbate peroxidase reactions does not result in a photobaric signal, as O₂-uptake and O₂-evolution components cancel each other. Two main conclusions, which are of considerable importance for future practical applications of the photoacoustic method, are drawn from these findings: (1) When high CO₂ is applied to leaves, the photobaric uptake component may provide a unique means of monitoring the function of stromal carbonic anhydrase in vivo. (2) Brief flushing of the photoacoustic cell with air may prevent the occurrence of an uptake signal, thus allowing a straight-forward deconvolution into photothermal and O₂-evolution components.     

Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior pituitary cells.

We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.     

Kritische Untersuchungen zum histochemischen Nachweis der Phosphatasen sowie deren Beeinflussbarkeit in vitro und in vivo

Zusammenfassung Anhand umfangreicher Untersuchungen wird gezeigt, in welchem Maße die Ergebnisse des histochemischen Nachweises der Phosphatasen von der Methode abhängig sind. Es wird weiterhin auf die unterschiedliche Wirkung von SH-Gruppen-Hemmstoffen auf die Phosphatasen in vivo und in vitro eingegangen. Abschließend wird als Ergebnis dieser Untersuchungen eine Methode empfohlen, die es erlaubt, die ATP-ase, AMP-ase und GP-ase histochemisch optimal darzustellen und gleichzeitig auch die Wirkung verschiedenartiger Substanzen auf die Phosphatasen am Schnitt zu prüfen.